The present invention relates to methods for detecting the presence or concentration of a biological analyte in a matrix, such as urine or blood, by exploiting the specificities of an antibody and a signal-generating substrate for the analyte. More particularly, the present invention relates to methods for detecting the presence or concentration of an analyte, such as hemoglobin or a transferase or a serine protease, in a matrix by exploiting the antibody specific to the analyte to extract the analyte from its matrix, and by exploiting the substrate specific to the enzymatic property of the analyte in order to generate a signal proportionate to the amount of analyte in the matrix.
The known in vitro methods for directly detecting the presence or concentration of an analyte in a matrix using its specificity for its substrate have several disadvantages. Often, for instance, the matrix may contain substances which may interfere or inhibit direct testing for the analyte, thereby giving false positive or negative indications. Also, if the matrix is slightly hemolyzed, it may interfere with the measurement of light absorbance during spectrophotometric analysis. Likewise, any turbidity in the matrix will also obstruct the measurement of light absorbance during spectrophotometric analysis.
The known methods for testing hemoglobin in a matrix readily identify the disadvantages of such techniques. In particular, since hemoglobin acts as a pseudoperoxidase enzyme, known methods for directly testing a sample for hemoglobin have utilized hydrogen peroxide as a substrate. If hemoglobin is present in the matrix, it will liberate from hydrogen peroxide nascent oxygen which in turn oxidizes an added colorless dye into a colored form. However, the matrix being tested for hemoglobin may contain interfering substances, such as urine containing vitamin C or glutathione or their metabolites which may inhibit oxidation of the colorless dye. Thus, a false negative result may be obtained. Also, the matrix may contain interfering substances which give the same reaction as hemoglobin. For example, a feces sample containing vegetable peroxidase may react like hemoglobin when directly tested by the use of a peroxide substrate and colorless dye. Accordingly, in this situation, a false positive indication for the presence of hemoglobin may be obtained.
The presence of hemoglobin or its isoforms in a matrix has also been determined by various immunoassays known in the art. One such method captures the hemoglobin molecules by binding them to an antibody attached to a solid surface, and then enumerates the molecules by binding them with yet another antibody that is enzyme labeled. Alternatively, in other known methods, a second antibody is bound to the captured hemoglobin and is enumerated by an enzyme-labeled, third antibody. Thus, these types of immunoassays ignore the specific substrate for the enzymatic, or pseudoperoxidase property of the hemoglobin molecule and instead require the need for a secondary (or tertiary) antibody label.
Similar problems and disadvantages are encountered with the known methods used to test for gamma-glutamyl transferase enzyme (or "GGT"). For example, known methods for directly testing a matrix for GGT by utilizing a signal-generating specific substrate are also subject to interferences by other substances in the matrix. Substances such as citrate, oxalate, and fluoride are known to suppress GGT enzyme activity, thereby giving falsely low values in the measurement of light absorbance in a spectrophotometer. In addition, if the sample or matrix is slightly hemolyzed, it may absorb light in the same or close wavelength range as the GGT-substrate signal, thereby artificially increasing light absorbance and giving a false positive value.
Accordingly, there has existed a definite need for a method of testing for the presence or concentration of an analyte in a matrix which overcomes these disadvantages. Specifically, there is a need for a method which exploits the antibody specificity of the analyte to avoid the possible interferences of other substances contained within the matrix, and which exploits the substrate specificity of the enzymatic property of the analyte to generate a signal to indicate the analyte's presence or concentration. The present invention satisfies these needs and provides further related advantages.